1. Field of the Invention
The present invention relates to a method for amplifying an unknown nucleotide sequence adjacent to a known nucleotide sequence, in particular to a method for amplifying an unknown nucleotide sequence using uniquely designed primers and its applications.
2. Description of the Related Art
The polymerase chain reaction (PCR) presents the most effective method for selectively amplifying specific DNA fragments. In the PCR procedure, oligonucloetides complementary to the known 5′ and 3′ sequences flanking the target nucleic acid serve as “primers” and play a key role.
Application of PCR to isolate and analyze a particular DNA region requires knowledge of the DNA sequences flanking the region of interest. This generally limits amplification to regions of known DNA sequence. In the absence of the necessary sequence information, PCR amplification of a target DNA fraction in a complex DNA population is likely to result in the amplification of non-target DNA.
Many PCR-based methods have been developed and modified to isolate an unknown DNA sequence that flanks regions of known sequences. They include, inverse PCR (Triglia et al., 1988), panhandle PCR (Shyamala et al., 1989; Jones and Winistorfer, 1997), vectorette PCR (Arnold et al., 1991), anchored PCR (Roux et al., 1990), AP-PCR (Dominguez et al., 1994; Trueba and Johnson, 1996), capture PCR (Lagerstrom et al., 1991), and adapter- or cassette-ligated PCR (Iwahana et al., 1994; Riley et al., 1990; Siebert et al., 1995; Willems, 1998; Kilstrup and Kristiansen, 2000).
However, these methods have limitations such as the need to digest the DNA with restriction enzymes, ligate the digested DNA with linkers or with double-stranded, partially double-stranded, or single-stranded oligonucleotide cassettes, and purify and/or subclone the products before sequencing. The need for multiple steps in these protocols makes them cumbersome and inefficient. Furthermore, in these methods, the common problem is high background and non-specific products due to non-specific binding of the vector, adaptor, cassette, or tail primers.
Therefore, the methodology was further improved to provide a biotin/streptavidin system to capture biotinylated fragments of interest before the nested PCR is carried out (Rosenthal and Jones, 1990; Mishra et al. 2002). This method show an improvement to reduce the noise and to allow the amplification of the flanking region of any known sequence but requires a complicated procedure of immobilized step and also a lot of cost.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.